A convenient dry culture device for growing microorganisms is described in U.S. Pat. No. 4,565,783 to Hansen et al. In a typical device reported by Hansen et at., a cold-water soluble dry powder containing a gelling agent and microbial growth nutrients is coated on a waterproof substrate. A transparent, read-through cover sheet coated on a surface with an acrylate adhesive containing an indicating dye is attached to the coated substrate. When the device is used, a predetermined amount of an aqueous sample is typically placed in contact with the coated substrate and the cover sheet is placed over the sample and substrate. The aqueous sample hydrates the soluble dry powder which then forms a gelled medium capable of sustaining microbial growth. During the growth period, the indicator dye adhered to the cover sheet reacts in the presence of viable microorganisms to give a detectable response that allows visualization of bacterial colonies which are grown on the culture device. A dry culture device based on the report of Hansen et al. is commercially available as Aerobic Count PETRIFILM plates (Catalog No. 6400, 3M, St. Paul, Minn.).
Another convenient dry culture device for growing aerobic microorganisms such as yeasts and molds is described in U.S. Pat. No. 5,089,413 to Nelson et at. Like the device of Hansen et al., the Nelson et al. device includes a cold-water soluble dry powder containing a gelling agent and microbial growth nutrients coated on a substrate as well as a transparent cover sheet coated with an acrylate adhesive containing an indicating dye. The Nelson et al. device differs from the Hansen et al. device because it contains an additional air permeable membrane layer in order to provide sufficient air or oxygen transmission which is needed for the growth of aerobic organisms such as yeasts and molds. In addition, the nutrients are selected for the growth of yeasts and molds, the indicator dyes are generally specific for yeasts and molds, and antibacterial agents are added to the nutrients in order to prevent contamination of the devices by bacteria or other unwanted microorganisms.
The dry culture devices of both Hansen et al. and Nelson et al. are much simpler to use than conventional gelled agar medium/petri dish systems because there is no need for the user to heat and mix the growth medium, agar and other reagents and then add the mixture to the pour plates. In addition, the devices of Hansen et al. and Nelson et al. are compact and easily disposed of and therefore are easier and safer to use.
In spite of the many advantages that the Hansen et al. or Nelson et al. devices have over conventional types of culture systems, there is a need for increased reagent stability and shelf life in the dry culture devices described above. For example, it has been observed that certain indicator dyes that may be used to aid in the visualization of bacterial colonies grown using the Hansen et al. system tend to be unstable over a period of time in the presence of the other components of the system. See, for example, U.S. Pat. No. 3,867,258 to Forgione that reports that tetrazolium salts used in diagnostic test devices may be stabilized with selected, known antioxidants including alkylated phenols such as butylated hydroxy toluene (BHT), thiobisphenols such as 4,4'-thiobis(6-t-butyl-3-methylphenol) or esters such as distearyl thiodipropionate. In addition, it has been observed that reduction sensitive indicators such as tetrazolium salts, when adhered to thin films with a water-insoluble pressure sensitive adhesive, may inexplicably be reduced to the colored form of the indicator when stored.
Thus, a need exists in the art for a dry culture system which has improved shelf life and enhanced indicator stability compared to current devices.